A systematic analysis of the structure, function, and regulation of the pyruvate and alpha-ketogluterate dehydrogenase complexes from microorganisms and from mammalian tissues is continuing. Activity of the mammalian pyruvate dehydrogenase complex is regulated by phosphorylation and dephosphorylation catalyzed, respectively, by a MgATP2-requiring kinase and a Mg2 ion-requiring phosphatase. The site of this covalent regulation is the pyruvate dehydrogenase component of the complex. Pyruvate dehydrogenase exists as a tetramer, alpha 2 beta 2. Phosphorylation occurs on seryl residues in the alpha-chain, thereby inactivating the enzyme. It appears that phosphorylation produces a conformational change in pyruvate dehydrogenase that displaces a catalytic group (or groups) at the active center. We have obtained evidence that phosphopeptides produced by tryptic digestion of phosphorylated pyruvate dehydrogenase are effective substrates for pyruvate dehydrogenase phosphatase and that the dephosphopeptides can serve as substrates for pyruvate dehydrogenase kinase. These findings indicate that the phosphatase and the kinase do not require an intact tertiary structure in pyruvate dehydrogenase, but apparently recognize components of the local primary sequence around the phosphorylation sites. A systematic study is being conducted with these peptide substrates to elucidate the site and mechanism of action of the known modifiers of pyruvate dehydrogenase kinase and phosphatase activities. BIBLIOGRAPHIC REFERENCES: Butler, J. R., Pettit, F. H., Davis, P. F., and Reed, L. J., "Binding of Thiamin Thiazolone Pyrophosphate to Mammalian Pyruvate Dehydrogenase and Its Effects on Kinase and Phosphatase Activities," Biochem. Biophys. Res. Commun., 74, 1667-1674 (1977). Davis, P. F., Pettit, F. H., and Reed, L. J., "Peptides Derived from Pyruvate Dehydrogenase as Substrates for Pyruvate Dehydrogenase Kinase and Phosphatase," Biochem. Biophys. Res. Commun., 75, 541-549 (1977).